Breast cancer outcome has improved significantly over the last 10 years. However, a significant number of patients relapse despite current approaches, and a number of patients receive un-needed therapy as we are unable to distinguish which patients have low risk or high risk disease. Breast cancer behavior is regulated, in part, by 4 important protein signaling pathways (PI3K/PTEN/AKT, JAK/STAT, TGF-beta/Smad and MAPK/ERK1/2). They interact bidirectionally with hormone receptors and further interact at multiple levels creating a complex signaling network. Our hypothesis is that a comprehensive analysis of signaling events in breast cancer will identify patients likely to respond to particular therapeutic approaches. Our specific aims are: (1) To classify breast cancer by characterizing the functional proteomic expression/activation signature of four signal transduction cascades: PI3K/PTEN/AKT, JAK/STAT, TGF-beta/Smad and MAPK/ERK1/2, (2) to determine whether the functional proteomics based classification will predict response to preoperative chemotherapy, and (3) to correlate the functional proteomics based classification with gene expression signatures. We will perform reverse phase protein microarray (RPPA) in 90 frozen preoperative breast cancer samples and analyze the 4 signal transduction cascades. The data will be used as a training set to define a breast cancer signaling profile associated with response to preoperative chemotherapy. We will obtain a validation set of 50 samples and apply the proteomic profile to determine its specificity and sensitivity in predicting response to therapy. We will also correlate the predictive value of the functional proteomics data with that of gene expression. There is a crucial need for approaches which will predict which breast cancers will respond to therapy. As proteins are the direct executors of cell function, and since RPPA can concurrently assess total protein levels, protein phosphorylation, cell cycle, apoptosis and neovascularization, this technology may reflect tumor biology more acurately. A comprehensive assessment of the molecular mechanisms of carcinogenesis and cell survival obviously involves the evaluation of both the genome and proteome. The research in this proposal will lead to a better understanding of the mechanisms underlying the wide variation in breast cancer behavior and responsiveness to therapy and has potential to identify other therapeutic targets in patients for whom conventional treatment is inadequate. [unreadable] [unreadable] [unreadable]